The steroid hormone estrogen plays a critical role in the development and maintenance of female reproductive and mammary tissues, but is also involved in cardiovascular, skeletal, and neural cell function. Estrogens and selective estrogen receptor modulators are widely used in regulating fertility, alleviating postmenopausal symptoms, and preventing and treating breast cancer. The biological effects of these hormones are initiated by binding to the estrogen receptor (ER) and inducing the receptor to bind to estrogen response elements (EREs) residing in target genes. It is this interaction of the receptor with the ERE, in cooperation with multiple coregulatory proteins, that leads to changes in gene expression. During the previous award period, we isolated and identified more than 100 proteins that associate with the DNA-bound ERa and showed that a number of these ERa-associated proteins interact with the receptor, increase the interaction of the receptor with DNA, and influence receptor-mediated gene expression. Interestingly, rather than functioning individually, these proteins form integrated networks endowed with a variety of enzymatic and catalytic functions. A number of the proteins we identified are involved in responding to oxidative stress, which has been linked to aging and human disease. Oxidative stress proteins play pivotal roles in maintaining normal cell function. Using molecular, cell-based, and in vivo approaches we will (1) determine whether estrogen increases expression of the oxidative stress proteins Cu/Zn superoxide dismutase (SOD1), thioredoxin (Trx), thioredoxin reductase (TrxR), protein disulfide isomerase (PDI), and apurinic endonuclease 1 (APE1) in the cerebral cortex of C57BL/6 mice, (2) define whether estrogens and/or progestins alter the expression of oxidative stress proteins in the cerebral cortex using organotypic brain slice cultures, (3) delineate whether hormone-induced expression of oxidative stress proteins plays a role in protecting the cerebral cortex from ischemia and identify the individual oxidative stress proteins involved in this neuroprotection, and (4) define the role of oxidative stress proteins in estrogen-responsive gene expression. The insights gained will help delineate how estrogens and other hormonal ligands regulate gene expression in the brain, enhance our understanding of the cellular responsiveness of the brain to an array of clinically important pharmaceutical agents, and define hormonal treatments that may help to protect the brain from ischemic insults such as stroke. PUBLIC HEALTH RELEVANCE: It is well established that estrogen protects the brain from damage when the blood supply has been interrupted as occurs during stroke. However, the mechanisms by which estrogen confers this protective effect are unknown. We have substantial preliminary evidence to suggest that estrogen induces the expression of specific proteins involved in this neuroprotection. We will first determine whether estrogen induces expression of 5 specific oxidative stress proteins in the brain and then determine whether these proteins are involved in protecting the brain from ischemia. Our studies will provide critical new information about the role of a variety of estrogens and progestins in the brain and aid in the design of more effective hormonal treatments for women of all ages.